The neurotoxins of Clostridium botulinum (BoNTs) are the most potent protein toxins for humans and are included in the list of Category A Select Agents and Toxins (14). BoNTs comprise seven distinguishable serotypes, A-G, with serotypes A, B, and E responsible for most natural human intoxications (19). Each BoNT serotype is classically defined by the specificity of antibody neutralization. Thus, antibodies that neutralize BoNT serotype A (BoNT/A) do not neutralize the toxicity of BoNT serotypes B-G.
Currently available vaccines are composed of chemically inactivated crude isolates of BoNTs. There are two available therapies against botulism, a pentavalent vaccine against serotypes A-E (20) and a heptavalent immune globulin against serotypes A-G (27). However, these vaccines are produced from chemically inactivated BoNT that is produced in C. botulinum and is currently in limited supply. There is a need to develop more efficient approaches for vaccine development against botulism.
BoNTs are zinc proteases that elicit flaccid paralysis by inhibiting the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. BoNTs are produced as ˜150 kDa nontoxic single chain proteins that are activated by proteolytic cleavage to a dichain structure. BoNTs comprise three functional domains, organized as an N-terminal catalytic domain (Light Chain, LC), an internal translocation domain (Heavy Chain, HCT), and a C-terminal receptor binding domain (Heavy Chain, HCR) (FIG. 1A). In addition, HCR can be divided into a N-terminal domain (HCRN) and a C-terminal domain (HCRC).
HCRC has been implicated to possess receptor binding capacity for neurons (23). BoNTs enter neurons via receptor-mediated endocytosis. The neurotoxicity of BoNTs is due to the affinity of HCR for protein(s) on the plasma membrane of peripheral neurons (22). The HCR-plasma membrane receptor interaction is enhanced by gangliosides, which are low affinity co-receptors for HCR (12). The translocation capabilities of HCT have been extrapolated from the action of the translocation domain of diphtheria toxin (8). Both native and recombinant HC form channels in artificial lipid bilayers through which the LC can be translocated (17). Upon delivery into the cytosol, LC cleaves neurotransmitter vesicle docking proteins, BoNT/A cleaves SNAP25 between residues 197-198 and BoNT/E cleaves SNAP25 between residues 180-181, which inactivates SNAP25 (33).
In addition to the 7 serotypes of BoNT (A-G) (13, 16) several BoNT variants (sub-serotypes) have been identified that are immunologically distinguishable within a serotype. The classical type A-Hall strain (ATCC 3502) (BoNT/A1) and the Kyoto F infant strain (BoNT/A2) differ by ˜10% in their primary amino acid sequence (10, 11, 15), while BoNT/EB and BoNT/BA possess ˜92% primary amino acid homology.
New vaccine strategies for botulism based upon recombinant antigens are currently under development. Native and recombinant HCR purified from C. botulinum and Escherichia coil protect mice against BoNT/A challenge when administered intra-parenterally (i.p.) (29, 32). Currently the HCR domains of the BoNTs are being expressed in the yeast Pichia pastoris (26). While useful as a first generation recombinant BoNT vaccine, this approach has several limitations, including limited genetic manipulation (26).